Compositions, methods, systems and kits for target nucleic acid enrichment

ABSTRACT

The present invention provides methods, compositions, kits, systems and apparatus that are useful for isolating nucleic acid molecules from a sample. In particular, the methods generally relate to normalizing the concentration of target nucleic acid molecules from a sample. In one aspect, the invention relates to purifying a primer extension product from a primer extension reaction mixture. In some aspects, nucleic acid molecules obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. non-provisionalapplication Ser. No. 14/054,618, filed Oct. 15, 2013, now U.S. Pat. No.9,133,510, Issued Sep. 15, 2015, which claims benefit of priority under35 U.S.C. § 119(e) to U.S. Provisional Application No. 61/714,206, filedOct. 15, 2012 and U.S. Provisional Application No. 61/764,122 filed Feb.13, 2013 entitled “COMPOSITIONS, METHODS, SYSTEMS AND KITS FOR TARGETNUCLEIC ACID ENRICHMENT”, the disclosures of which are incorporatedherein by reference in their entireties.

All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, and treatises are expressly incorporated by reference in theirentirety for any purpose.

TECHNICAL FIELD

In some embodiments, the present teachings provide compositions,systems, methods, apparatuses and kits for enrichment of target nucleicacid molecules from a sample or reaction mixture.

BACKGROUND

Sample preparation involving enrichment of target nucleic acid moleculesfrom samples or reaction mixtures is frequently required prior todownstream applications such as cloning and nucleic acid sequencing.Typically, such downstream applications are performed in high throughputformat, increasing the labor, time, and reagent costs for samplepreparation prior to such techniques. Such applications frequentlyrequire the starting amounts and/or concentrations of nucleic acidmolecule sample inputs to be normalized (or standardized) within anoptimal working range. For example, many applications requirenormalization of nucleic acid samples before analysis can be performed,the purpose of such normalization being to substantially equalize thenumber of nucleic acid molecules (or concentration of nucleic acidmolecules) within each sample to each other. These steps ofquantification and normalization are extremely time-consuming andtedious; and strain laboratory resources as the number of target nucleicacid libraries to be quantified and/or normalized increases. Typically,to quantify a target nucleic acid library an aliquot of each sample isdiluted and the nucleic acid concentration is determined. If theconcentration of either or both samples varies significantly from theacceptable working range, the samples can be diluted or otherwiseadjusted to acceptable starting amounts or concentrations. In someinstances, the nucleic acid concentrations are adjusted to besubstantially equal to each other during the normalization process. Thisprocess is referred to as “normalization” resulting in the generation ofnormalized samples having substantially equal numbers (orconcentrations) of nucleic acid molecules. Such quantification and/ornormalization processes, in addition to being labor-intensive, alsoimpede the speed by which other downstream process can be initiated. Insome instances, the time required to quantify and normalize severalthousand target nucleic acid libraries can ultimately influence thespeed by which sequencing data can be obtained from such downstreamprocesses. Therefore, what is needed is an improved method fornormalizing the starting number and/or concentration of nucleic acidmolecules within one or more samples. What is also needed is a method bywhich to purify an extended primer product from a primer extensionreaction mixture. Further, a method for isolating a specific amount ofnucleic acid from a sample is desired.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic depicting a non-limiting embodiment of a targetnucleic acid molecule enrichment method.

FIG. 2 is a schematic depicting exemplary results obtained using anon-limiting embodiment of the target nucleic acid molecule enrichmentmethod.

FIG. 3 is schematic depicting exemplary results obtained using anon-limiting embodiment of the target nucleic acid molecule enrichmentmethod.

SUMMARY

One particular exemplary application that can benefit from use of thetarget enrichment methods and normalization methods disclosed herein isnucleic acid sequencing, including next-generation sequencing (NGS).Many NGS platforms including the Ion Torrent Sequencers (Personal GenomeMachine™ and Ion Torrent Proton™ Sequencers (Life Technologies, CA)require advance preparation and enrichment of large numbers of targetnucleic acid molecules to be sequenced. Further details regarding thecompositions, design and operation of the Ion Torrent PGM™ sequencer canbe found, for example, in U.S. patent application Ser. No. 12/002,781,now published as U.S. Patent Publication No. 2009/0026082; U.S. patentapplication Ser. No. 12/474,897, now published as U.S. PatentPublication No. 2010/0137143; and U.S. patent application Ser. No.12/492,844, now published as U.S. Patent Publication No. 2010/0282617,all of which applications are incorporated by reference herein in theirentireties. Various library preparation methods and kits exist withinthe NGS field that allow for the preparation of multiple target nucleicacid molecules from a single source (Ion Ampliseq™ Library Preparation,Publication Part Number: MAN0006735 or Ion Xpress™ Plus gDNA FragmentLibrary Preparation, Publication Part Number 4471989 (Life Technologies,CA); NEBNEXT® Fast DNA Library Prep Set for Ion Torrent, New EnglandBiolabs Catalog # E6270L). The advent of barcoding has expanded thisfunctionality by allowing the indexing of multiple target nucleic acidmolecules from multiple samples or sources in a single sequencing run(Ion Xpress™ Barcode Adaptors 1-96 for use with Ion Xpress™ PlusFragment Library Kit (Life Technologies, CA); Access Array™ BarcodeLibrary, Fluidigm Corp, CA). Some areas of NGS, such as targetedre-sequencing, typically utilize many samples prepared in parallel, forexample in several 96-well plates. The starting amounts of barcoded andnon-barcoded nucleic acid libraries prepared using known librarypreparation methods vary widely and thus must be individually quantifiedbefore being transitioned into downstream processes. Quantification oftarget nucleic acid libraries can be achieved using a variety ofprotocols, including qPCR, Qubit® Fluorometer (Life Technologies, CA)and Bioanalyzer™ (Agilent Technologies, CA).

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the described subject matter inany way.

All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and internet web pages are expressly incorporated byreference in their entirety for any purpose. When definitions of termsin incorporated references appear to differ from the definitionsprovided in the present teachings, the definition provided in thepresent teachings shall control.

It will be appreciated that there is an implied “about” prior to thetemperatures, concentrations, times, etc discussed in the presentteachings, such that slight and insubstantial deviations are within thescope of the present teachings herein.

Unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”,“containing”, “include”, “includes”, and “including” are not intended tobe limiting.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention.

Unless otherwise defined, scientific and technical terms used inconnection with the present teachings described herein shall have themeanings that are commonly understood by those of ordinary skill in theart. Generally, nomenclatures utilized in connection with, andtechniques of, cell and tissue culture, molecular biology, and proteinand oligo- or polynucleotide chemistry and hybridization describedherein are those well known and commonly used in the art. Standardtechniques are used, for example, for nucleic acid purification andpreparation, chemical analysis, recombinant nucleic acid, andoligonucleotide synthesis. Enzymatic reactions and purificationtechniques are performed according to manufacturer's specifications oras commonly accomplished in the art or as described herein. Thetechniques and procedures described herein are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the instant specification. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual (Third ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). Thenomenclatures utilized in connection with, and the laboratory proceduresand techniques described herein are those well known and commonly usedin the art.

As utilized in accordance with exemplary embodiments provided herein,the following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein the term “amplification” and its variants includes anyprocess for producing multiple copies or complements of at least someportion of a polynucleotide, said polynucleotide typically beingreferred to as a “template” or, in some cases, as a “target.” Thetemplate (or target) polynucleotide can be single stranded or doublestranded. Amplification of a given template can result in the generationof a population of polynucleotide amplification products, collectivelyreferred to as an “amplicon”.

As used herein, the term “sequencing” and its variants compriseobtaining sequence information from a nucleic acid strand, typically bydetermining the identity of at least one nucleotide (including itsnucleobase component) within the nucleic acid strand. While in someembodiments, “sequencing” a given region of a nucleic acid moleculeincludes identifying each and every nucleotide within the region that issequenced, “sequencing” can also include methods whereby the identity ofone or more nucleotides in is determined, while the identity of somenucleotides remains undetermined or incorrectly determined.

The terms “identity” and “identical” and their variants, as used herein,when used in reference to two or more nucleic acid sequences, refer tosimilarity in sequence of the two or more sequences (e.g., nucleotide orpolypeptide sequences). In the context of two or more homologoussequences, the percent identity or homology of the sequences orsubsequences thereof indicates the percentage of all monomeric units(e.g., nucleotides or amino acids) that are the same (i.e., about 70%identity, preferably 75%, 80%, 85%, 90%, 95% or 99% identity). Thepercent identity can be over a specified region, when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection. Sequences are said to be“substantially identical” when there is at least 85% identity at theamino acid level or at the nucleotide level. Preferably, the identityexists over a region that is at least about 25, 50, or 100 residues inlength, or across the entire length of at least one compared sequence. Atypical algorithm for determining percent sequence identity and sequencesimilarity are the BLAST and BLAST 2.0 algorithms, which are describedin Altschul et al, Nuc. Acids Res. 25:3389-3402 (1977). Other methodsinclude the algorithms of Smith & Waterman, Adv. Appl. Math. 2:482(1981), and Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), etc.Another indication that two nucleic acid sequences are substantiallyidentical is that the two molecules or their complements hybridize toeach other under stringent hybridization conditions.

The term “complementary” and its variants, as used herein in referenceto two or more polynucleotides or nucleic acid sequences, refer topolynucleotides (or sequences within one or more polynucleoties)including any nucleic acid sequences that can undergo cumulative basepairing at two or more individual corresponding positions inantiparallel orientation, as in a hybridized duplex. Optionally therecan be “complete” or “total” complementarity between a first and secondnucleic acid sequence where each nucleotide in one of the nucleic acidsequences can undergo a stabilizing base pairing interaction with anucleotide in the corresponding antiparallel position in the othernucleic acid sequence (however, the term “complementary” by itself caninclude nucleic acid sequences that include some noncomplementaryportions, for example when one nucleic acid sequence is longer than theother). “Partial” complementarity describes nucleic acid sequences inwhich at least 20%, but less than 100%, of the residues of one nucleicacid sequence are complementary to residues in the other nucleic acidsequence. In some embodiments, at least 50%, but less than 100%, of theresidues of one nucleic acid sequence are complementary to residues inthe other nucleic acid sequence. In some embodiments, at least 70%, 80%,90% or 95%, but less than 100%, of the residues of one nucleic acidsequence are complementary to residues in the other nucleic acidsequence. Sequences are said to be “substantially complementary” when atleast 80% of the residues of one nucleic acid sequence are complementaryto residues in the other nucleic acid sequence. “Noncomplementary”describes nucleic acid sequences in which less than 20% of the residuesof one nucleic acid sequence are complementary to residues in the othernucleic acid sequence. A “mismatch” is present at any position in thetwo opposed nucleotides are not complementary. Complementary nucleotidesinclude nucleotides that are efficiently incorporated by DNA polymerasesopposite each other during DNA replication under physiologicalconditions. In a typical embodiment, complementary nucleotides can formbase pairs with each other, such as the A-T/U and G-C base pairs formedthrough specific Watson-Crick type hydrogen bonding between thenucleobases of nucleotides and/or polynucleotides at positionsantiparallel to each other. The complementarity of other artificial basepairs can be based on other types of hydrogen bonding and/orhydrophobicity of bases and/or shape complementarity between bases.

The term “hybridize” or “anneal” and their variants, as used herein inreference to two or more polynucleotides, refer to any process wherebyany one or more nucleic acid sequences (each sequence comprising astretch of contiguous nucleotide residues) within said polynucleotidesundergo base pairing at two or more individual corresponding positions,for example as in a hybridized nucleic acid duplex. Optionally there canbe “complete” or “total” hybridization between a first and secondnucleic acid sequence, where each nucleotide residue in the firstnucleic acid sequence can undergo a base pairing interaction with acorresponding nucleotide in the antiparallel position on the secondnucleic acid sequence. In some embodiments, hybridization can includebase pairing between two or more nucleic acid sequences that are notcompletely complementary, or are not base paired, over their entirelength. For example, “partial” hybridization occurs when two nucleicacid sequences undergo base pairing, where at least 20% but less than100%, of the residues of one nucleic acid sequence are base paired toresidues in the other nucleic acid sequence. In some embodiments,hybridization includes base pairing between two nucleic acid sequences,where at least 50%, but less than 100%, of the residues of one nucleicacid sequence are base paired with corresponding residues in the othernucleic acid sequence. In some embodiments, at least 70%, 80%, 90% or95%, but less than 100%, of the residues of one nucleic acid sequenceare base paired with corresponding residues in the other nucleic acidsequence. Two nucleic acid sequences are said to be “substantiallyhybridized” when at least 85% of the residues of one nucleic acidsequence are base paired with corresponding residues in the othernucleic acid sequence. In situations where one nucleic acid molecule issubstantially longer than the other (or where the two nucleic acidmolecule include both substantially complementary and substantiallynon-complementary regions), the two nucleic acid molecules can bedescribed as “hybridized” even when portions of either or both nucleicacid molecule can remain unhybridized. “Unhybridized” describes nucleicacid sequences in which less than 20% of the residues of one nucleicacid sequence are base paired to residues in the other nucleic acidsequence. In some embodiments, base pairing can occur according to someconventional pairing paradigm, such as the A-T/U and G-C base pairsformed through specific Watson-Crick type hydrogen bonding between thenucleobases of nucleotides and/or polynucleotides positions antiparallelto each other; in other embodiments, base pairing can occur through anyother paradigm whereby base pairing proceeds according to establishedand predictable rules.

Hybridization of two or more polynucleotides can occur whenever said twoor more polynucleotides come into contact under suitable hybridizingconditions. Hybridizing conditions include any conditions that aresuitable for nucleic acid hybridization; methods of performinghybridization and suitable conditions for hybridization are well knownin the art. The stringency of hybridization can be influenced by variousparameters, including degree of identity and/or complementarity betweenthe polynucleotides (or any target sequences within the polynucleotides)to be hybridized; melting point of the polynucleotides and/or targetsequences to be hybridized, referred to as “T_(m)”; parameters such assalts, buffers, pH, temperature, GC % content of the polynucleotide andprimers, and/or time. Typically, hybridization is favored in lowertemperatures and/or increased salt concentrations, as well as reducedconcentrations of organic solvents. High-stringency hybridizationconditions will typically require a higher degree of complementarybetween two target sequences for hybridization to occur, whereaslow-stringency hybridization conditions will favor hybridization evenwhen the two polynucleotides to be hybridized exhibit lower levels ofcomplementarity. The hybridization conditions can be applied during ahybridization step, or an optional and successive wash step, or both thehybridization and optional wash steps.

Examples of high-stringency hybridization conditions include any one ormore of the following: salt concentrations (e.g., NaCl) of from about0.0165 to about 0.0330 M; temperatures of from about 5° C. to about 10°C. below the melting point (T_(m)) of the target sequences (orpolynucleotides) to be hybridized; and/or formamide concentrations ofabout 50% or higher. Typically, high-stringency hybridization conditionspermit binding between sequences having high homology, e.g., ≥95%identity or complementarity. In one exemplary embodiment ofhigh-stringency hybridization conditions, hybridization is performed atabout 42° C. in a hybridization solution containing 25 mM KPO₄ (pH 7.4),5×SSC, 5×Denhardt's solution, 50 μg/mL denatured, sonicated salmon spermDNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/mL double strandedpolynucleotide (or double stranded target sequence), while the washesare performed at about 65° C. with a wash solution containing 0.2×SSCand 0.1% sodium dodecyl sulfate.

Examples of medium-stringency hybridization conditions can include anyone or more of the following: salt concentrations (e.g., NaCl) of fromabout 0.165 to about 0.330 M; temperatures of from about 20° C. to about29° C. below the melting point (T_(m)) of the target sequences to behybridized; and/or formamide concentrations of about 35% or lower.Typically, such medium-stringency conditions permit binding betweensequences having high or moderate homology, e.g., ≥80% identity orcomplementarity. In one exemplary embodiment of medium stringencyhybridization conditions, hybridization is performed at about 42° C. ina hybridization solution containing 25 mM KPO₄ (pH 7.4), 5×SSC, 5×Denhart's solution, 50 μg/mL denatured, sonicated salmon sperm DNA, 50%formamide, 10% Dextran sulfate, and 1-15 ng/mL double strandedpolynucleotide (or double stranded target sequence), while the washesare performed at about 50° C. with a wash solution containing 2×SSC and0.1% sodium dodecyl sulfate.

Examples of low-stringency hybridization conditions include any one ormore of the following: salt concentrations (e.g., NaCl) of from about0.330 to about 0.825 M; temperatures of from about 40° C. to about 48°C. below the melting point (T_(m)) of the target sequences to behybridized; and/or formamide concentrations of about 25% or lower.Typically, such low-stringency conditions permit binding betweensequences having low homology, e.g., ≥50% identity or complementarity.

Some exemplary conditions suitable for hybridization include incubationof the polynucleotides to be hybridized in solutions having sodiumsalts, such as NaCl, sodium citrate and/or sodium phosphate. In someembodiments, hybridization or wash solutions can include about 10-75%formamide and/or about 0.01-0.7% sodium dodecyl sulfate (SDS). In someembodiments, a hybridization solution can be a stringent hybridizationsolution which can include any combination of 50% formamide, 5×SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%sodium pyrophosphate, 5×Denhardt's solution, 0.1% SDS, and/or 10%dextran sulfate. In some embodiments, the hybridization or washingsolution can include BSA (bovine serum albumin). In some embodiments,hybridization or washing can be conducted at a temperature range ofabout 20-25° C., or about 25-30° C., or about 30-35° C., or about 35-40°C., or about 40-45° C., or about 45-50° C., or about 50-55° C., orhigher.

In some embodiments, hybridization or washing can be conducted for atime range of about 1-10 minutes, or about 10-20 minutes, or about 20-30minutes, or about 30-40 minutes, or about 40-50 minutes, or about 50-60minutes, or longer.

In some embodiments, hybridization or wash conditions can be conductedat a pH range of about 5-10, or about pH 6-9, or about pH 6.5-8, orabout pH 6.5-7.

As used herein, the terms “melting temperature” and “T_(m)” and theirvariants, when used in reference to a given polynucleotide (or a giventarget sequence within a polynucleotide) typically refers to atemperature at which 50% of the given polynucleotide (or given targetsequence) exists in double-stranded form and 50% is single stranded,under a defined set of conditions. In some embodiments, the defined setof conditions can include a defined parameter indicating ionic strengthand/or pH in an aqueous reaction condition. A defined condition can bemodulated by altering the concentration of salts (e.g., sodium),temperature, pH, buffers, and/or formamide. Typically, the calculatedthermal melting temperature can be at about 5-30° C. below the T_(m), orabout 5-25° C. below the T_(m), or about 5-20° C. below the T_(m), orabout 5-15° C. below the T_(m), or about 5-10° C. below the T_(m). TheTm of a given nucleic acid sequence can be calculated according to anysuitable method (including both actual melting assays as well as T_(m)prediction algorithms) as long as comparisons of T_(m) values areperformed using T_(m) values obtained using the same calculationmethods. Methods for calculating a T_(m) are well known and can be foundin Sambrook (1989 in “Molecular Cloning: A Laboratory Manual”, 2^(nd)edition, volumes 1-3; Wetmur 1966, J. Mol. Biol., 31:349-370; Wetmur1991 Critical Reviews in Biochemistry and Molecular Biology,26:227-259). Other sources for calculating a T_(m) for hybridizing ordenaturing nucleic acids include OligoAnalyze (from Integrated DNATechnologies), OligoCalc and Primer3 (distributed by the WhiteheadInstitute for Biomedical Research). In some embodiments of the methodsprovided herein, the T_(m) of a given nucleic acid sequence iscalculated according to any one or more of these methods. In someembodiments, the T_(m) is calculated assuming the following conditionsnucleic acid sequence is suspended in a solution including 50 nM ofnucleic acid sequence in a Tris-based buffer including 50 nM salt (e.g.,NaCl). In some embodiments, the Tm is calculated assuming 50 nM nucleicacid sequence in a solution including 0.5M NaCl. In some embodiments,the Tm is calculated assuming that the nucleic acid sequence is presentat a concentration of 100 pM in a buffer containing: 10 mM Tris pH 8.0;500 mM NaCl; 0.1 mM EDTA and 0.05% Tween-20.

As used herein, the term “primer” and its variants can include anysingle stranded nucleic acid molecule (regardless of length) that, oncehybridized to a complementary nucleic acid sequence, can prime nucleicacid synthesis. Typically, such nucleic acid synthesis occurs in atemplate-dependent fashion, and nucleotides are polymerized onto atleast one end of the primer during such nucleic acid synthesis. The term“primer extension” and its variants, as used herein, when used inreference to a given method, relates to any method for catalyzingnucleotide incorporation onto a terminal end of a nucleic acid molecule.Typically but not necessarily such nucleotide incorporation occurs in atemplate-dependent fashion. In some embodiments, the primer extensionactivity of a given polymerase can be quantified as the total number ofnucleotides incorporated (as measured by, e.g., radiometric or othersuitable assay) by a unit amount of polymerase (in moles) per unit time(seconds) under a particular set of reaction conditions.

As used herein, the term “hairpin”, when used in reference to anyoligonucleotide, primer, polynucleotide or nucleic acid molecule, refersto an oligonucleotide, primer, polynucleotide or nucleic acid moleculethat includes two nucleic acid sequences (referred to herein as a “firsthairpin sequence” and a “second hairpin sequence” that are at least 70%complementary to each other. In some embodiments, the first and secondhairpin sequences are at least 75%, 77%, 80%, 85%, 90%, 95%, 97%, 99%complementary to each other, or are complementary. The first and secondhairpin sequences are optionally capable of hybridizing to each otherunder suitable conditions. The hybrid formed via hybridization of firstand second hairpin sequences to each other can have a meltingtemperature (T_(m)) referred to as “the hairpin melting temperature” or“the hairpin T_(m)”. Typically, the first and second hairpin sequencesare in reverse orientation with respect to each other, such thathybridization of the first and second hairpin sequences will result inthe formation of a hairpin structure at temperatures below the hairpinT_(m). In some embodiments, the hairpin oligonucleotide, primer,polynucleotide or nucleic acid molecule exists predominantly in thehairpin form at temperatures significantly below the hairpin meltingtemperature, and predominantly in the extended (melted) single-strandedform at temperatures significantly above the hairpin meltingtemperature. The first and second hairpin sequences can be of anylength, but are typically greater than 4 nucleotides long, even moretypically greater than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,25, 50, 75 or 100 nucleotides long. In some embodiments, the meltingtemperature (Tm) of the first and second sequences is lower than about80° C., or lower than about 70° C., or lower than about 65° C., or lowerthan about 60° C., or lower than about 55° C. when measured understandard PCR extension conditions.

As used herein, the term “nucleotide” and its variants comprise anycompound that can bind selectively to, or can be polymerized by, apolymerase. Typically, but not necessarily, selective binding of thenucleotide to the polymerase is followed by polymerization of thenucleotide into a nucleic acid strand by the polymerase; occasionallyhowever the nucleotide may dissociate from the polymerase withoutbecoming incorporated into the nucleic acid strand, an event referred toherein as a “non-productive” event. Such nucleotides include not onlynaturally-occurring nucleotides but also any analogs, regardless oftheir structure, that can bind selectively to, or can be polymerized by,a polymerase. While naturally-occurring nucleotides typically comprisebase, sugar and phosphate moieties, the nucleotides of the disclosurecan include compounds lacking any one, some or all of such moieties. Insome embodiments, the nucleotide can optionally include a chain ofphosphorus atoms comprising three, four, five, six, seven, eight, nine,ten or more phosphorus atoms. In some embodiments, the phosphorus chaincan be attached to any carbon of a sugar ring, such as the 5′ carbon.The phosphorus chain can be linked to the sugar with an intervening O orS. In one embodiment, one or more phosphorus atoms in the chain can bepart of a phosphate group having P and O. In another embodiment, thephosphorus atoms in the chain can be linked together with intervening O,NH, S, methylene, substituted methylene, ethylene, substituted ethylene,CNH₂, C(O), C(CH₂), CH₂CH₂, or C(OH)CH₂R (where R can be a 4-pyridine or1-imidazole). In one embodiment, the phosphorus atoms in the chain canhave side groups having O, BH₃, or S. In the phosphorus chain, aphosphorus atom with a side group other than O can be a substitutedphosphate group. Some examples of nucleotide analogs are described inXu, U.S. Pat. No. 7,405,281. In some embodiments, the nucleotidecomprises a label (e.g., reporter moiety) and referred to herein as a“labeled nucleotide”; the label of the labeled nucleotide is referred toherein as a “nucleotide label”. In some embodiments, the label can be inthe form of a fluorescent dye attached to the terminal phosphate group,i.e., the phosphate group or substitute phosphate group most distal fromthe sugar. Some examples of nucleotides that can be used in thedisclosed methods and compositions include, but are not limited to,ribonucleotides, deoxyribonucleotides, modified ribonucleotides,modified deoxyribonucleotides, ribonucleotide polyphosphates,deoxyribonucleotide polyphosphates, modified ribonucleotidepolyphosphates, modified deoxyribonucleotide polyphosphates, peptidenucleotides, metallonucleosides, phosphonate nucleosides, and modifiedphosphate-sugar backbone nucleotides, analogs, derivatives, or variantsof the foregoing compounds, and the like. In some embodiments, thenucleotide can comprise non-oxygen moieties such as, for example, thio-or borano-moieties, in place of the oxygen moiety bridging the alphaphosphate and the sugar of the nucleotide, or the alpha and betaphosphates of the nucleotide, or the beta and gamma phosphates of thenucleotide, or between any other two phosphates of the nucleotide, orany combination thereof.

As used herein, the term “nucleotide incorporation” and its variantscomprise polymerization of one or more nucleotides to form a nucleicacid strand including at least two nucleotides linked to each other,typically but not necessarily via phosphodiester bonds, althoughalternative linkages may be possible in the context of particularnucleotide analogs.

The terms “oligonucleotide”, “polynucleotide” “nucleic acid molecule”and their variants, as used herein, can be used interchangeably to referto any polymers that include one or more polynucleotide regions, withoutregard to the respective lengths of such polymers. In some embodiments,such polymers can include non-polynucleotide regions as well. Suchpolymers have at least 2 ends, which for sake of convenience may bereferred to herein as the 5′ and the 3′ end, although such terminologydoes not limit the structure of the underlying ends. For example, a 3′end of a primer, oligonucleotide, polynucleotide or nucleic acidmolecule does not necessarily include a free hydroxyl group and insteadcan include any other chemical group that can interact or react with anincoming nucleotide during a nucleotide incorporation reaction. Unlessmade otherwise clear by the context, such oligonucleotide,polynucleotide or nucleic acid molecule can be double stranded or singlestranded. While in some embodiments, the oligonucleotide (or primer) isshorter than a corresponding polynucleotide template (or templatenucleic acid molecule), in some embodiments the oligonucleotide orprimer will not be shorter than a corresponding polynucleotide template(or template nucleic acid molecule).

As used herein, the term “adaptor” includes polynucleotides oroligonucleotides comprising DNA, RNA, chimeric RNA/DNA molecules, oranalogs thereof. In some embodiments, an adaptor can include one or moreribonucleoside residues. In some embodiments, an adaptor can besingle-stranded or double-stranded nucleic acids, or can includesingle-stranded and/or double-stranded portions. In some embodiments, anadaptor can have any structure, including linear, hairpin, forked, orstem-loop.

In some embodiments, an adaptor can have any length, including fewerthan 10 bases in length, or about 10-20 bases in length, or about 20-50bases in length, or about 50-100 bases in length, or longer.

In some embodiments, an adaptor can have any combination of blunt end(s)and/or sticky end(s). In some embodiments, at least one end of anadaptor can be compatible with at least one end of a nucleic acidfragment. In some embodiments, a compatible end of an adaptor can bejoined to a compatible end of a nucleic acid fragment. In someembodiments, an adaptor can have a 5′ or 3′ overhang end.

In some embodiments, an adaptor can have a 5′ or 3′ overhang tail. Insome embodiments, the tail can be any length, including 1-50 or morenucleotides in length.

In some embodiments, an adaptor can include an internal nick. In someembodiments, an adaptor can have at least one strand that lacks aterminal 5′ phosphate residue. In some embodiments, an adaptor lacking aterminal 5′ phosphate residue can be joined to a nucleic acid fragmentto introduce a nick at the junction between the adaptor and the nucleicacid fragment.

In some embodiments, an adaptor can include a nucleotide sequence thatis part of, or is complementary to, any portion of a primer, or to theentire sequence of a primer, present in the amplification reactionmixture, or any portion of a sequencing primer, or the entire sequenceof a sequencing primer, or any portion thereof.

In some embodiments, an adaptor can include degenerate sequences. Insome embodiments, an adaptor can include one or more inosine residues.In some embodiments, a barcode adaptor can include a uniquelyidentifiable sequence. In some embodiments, a barcode adaptor can beused for constructing multiplex nucleic acid libraries.

In some embodiments, an adaptor can include at least one scissilelinkage. In some embodiments, a scissile linkage can be susceptible tocleavage or degradation by an enzyme or chemical compound. In someembodiments, an adaptor can include at least one phosphorothiolate,phosphorothioate, and/or phosphoramidate linkage.

In some embodiments, an adaptor can include identification sequences. Insome embodiments, an identification sequences can be used for sorting ortracking. In some embodiments, an identification sequences can be aunique sequence (e.g., barcode sequence). In some embodiments, a barcodesequence can allow identification of a particular adaptor among amixture of different adaptors having different barcodes sequences. Forexample, a mixture can include 2, 3, 4, 5, 6, 7-10, 10-50, 50-100,100-200, 200-500, 500-1000, or more different adaptors having uniquebarcode sequences.

In some embodiments, an adaptor can include any type of restrictionenzyme recognition sequence, including type I, type II, type IIs, typeIIB, type III or type IV restriction enzyme recognition sequences.

In some embodiments, an adaptor can include a cell regulation sequences,including a promoter (inducible or constitutive), enhancers,transcription or translation initiation sequence, transcription ortranslation termination sequence, secretion signals, Kozak sequence,cellular protein binding sequence, and the like.

In some embodiments, the term “substantially equal” and its variants,when used in reference to two or more values, refers to any two or morevalues that are less than 10 times each other, typically less than 5times, even more typically less than 3 times of any other value, evenmore typically no greater than 2 times each other. For example, forpurposes of the present disclosure, values of 1 and 9 can be consideredto be substantially equal, but not values of 1 and 20. In someembodiments, the term “substantially equal” is used to refer to two ormore output values each indicating a number of molecules or a finalconcentration of molecules derived from a sample. In some embodiments,“substantially equal” refers to an output nucleic acid concentration oftarget nucleic acid molecules isolated from two or more samples; forexample, output concentrations of nucleic acid molecules recovered froma first and second sample are said to be “substantially equal” if theoutput concentration from the first sample is less than 10 times theoutput concentration from the second sample. In some embodiments,“substantially equal” includes any output nucleic acid concentrationsfrom two or more sample that are within one standard deviation of eachother. In some embodiments, “substantially equal” refers to the absolutenumber of nucleic acid molecules isolated from two or more samples. Insome embodiments, a first number of target nucleic acid molecules isisolated from a first sample, and a second number of target nucleic acidmolecules is isolated from a second sample, where the first numbervaries by no more than 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 200%,250%, 500%, 750%, 900%, or 990% from the second number, or vice versa.In some embodiments, “substantially equal” refers to the concentrationof nucleic acid molecules isolated from two or more samples, wherein theconcentration of nucleic acid molecules between the two or more samplesvaries by less than 5 times, more preferably by less than 3 times, evenmore typically no greater than 2 times of each other. In someembodiments, “substantially equal” refers to the number or concentrationof nucleic acid molecules in a second sample, that is within about 1%,2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% of the meanconcentration or mean number of nucleic acid molecules in a firstsample. In some embodiments, a substantially equal output nucleic acidconcentration can include any concentration of from about 1 pM to about1000 pM, for example about 10 pM, 20 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900pM or more. In some embodiments, substantially equal outputs can includeany outputs' within a range of about 2-fold, 3-fold, 4-fold or 5-fold.

As used herein, the term “binding partners” includes two molecules, orportions thereof, which have a specific binding affinity for one anotherand typically will bind to each other in preference to binding to othermolecules. Typically but not necessarily some or all of the structure ofone member of a specific binding pair is complementary to some or all ofthe structure possessed by the other member, with the two members beingable to bind together specifically by way of a bond between thecomplementary structures, optionally by virtue of multiple noncovalentattractions.

In some embodiments, molecules that function as binding partnersinclude: biotin (and its derivatives) and their binding partner avidinmoieties, streptavidin moieties (and their derivatives); His-tags whichbind with nickel, cobalt or copper; cysteine, histidine, or histidinepatch which bind Ni—NTA; maltose which binds with maltose bindingprotein (MBP); lectin-carbohydrate binding partners; calcium-calciumbinding protein (CBP); acetylcholine and receptor-acetylcholine; proteinA and binding partner anti-FLAG antibody; GST and binding partnerglutathione; uracil DNA glycosylase (UDG) and ugi (uracil-DNAglycosylase inhibitor) protein; antigen or epitope tags which bind toantibody or antibody fragments, particularly antigens such asdigoxigenin, fluorescein, dinitrophenol or bromodeoxyuridine and theirrespective antibodies; mouse immunoglobulin and goat anti-mouseimmunoglobulin; IgG bound and protein A; receptor-receptor agonist orreceptor antagonist; enzyme-enzyme cofactors; enzyme-enzyme inhibitors;and thyroxine-cortisol. Another binding partner for biotin can be abiotin-binding protein from chicken (Hytonen, et al., BMC StructuralBiology 7:8).

An avidin moiety can include an avidin protein, as well as anyderivatives, analogs and other non-native forms of avidin that can bindto biotin moieties. Other forms of avidin moieties include native andrecombinant avidin and streptavidin as well as derivatized molecules,e.g. nonglycosylated avidins, N-acyl avidins and truncatedstreptavidins. For example, avidin moiety includes deglycosylated formsof avidin, bacterial streptavidins produced by Streptomyces (e.g.,Streptomyces avidinii), truncated streptavidins, recombinant avidin andstreptavidin as well as to derivatives of native, deglycosylated andrecombinant avidin and of native, recombinant and truncatedstreptavidin, for example, N-acyl avidins, e.g., N-acetyl, N-phthalyland N-succinyl avidin, and the commercial products ExtrAvidin™,Captavidin™, Neutravidin™ and Neutralite Avidin™.

In some embodiments, the disclosure relates generally to methods forisolating target nucleic acid molecules using a capture moiety, as wellas related compositions, kits, systems and apparatuses. In someembodiments, the methods (and relating compositions, systems,apparatuses and kits) can involve contacting a sample including apopulation of target nucleic acid molecules with first number ofmolecules of a capture oligonucleotide. In some embodiments, the captureoligonucleotide is capable of selectively binding to any one, some orall members of the population of target nucleic acid molecules; suchselective binding optionally includes sequence-specific hybridizationbetween a sequence of the capture oligonucleotide and a sequence of thetarget nucleic acid molecules of the population. In some embodiments(and in contrast to many conventional methods of target enrichment usingcapture oligonucleotides), the first number of capture oligonucleotidemolecules that are contacted with the target nucleic acid molecules canbe a limiting number. By “limiting” it is meant that the number ofmolecules of capture oligonucleotide is significantly less than thenumber of target nucleic acid molecules within the sample that arecapable of binding to the capture oligonucleotide. (Such embodimentscontrast with many conventional methods of enrichment for target nucleicacid molecules using capture oligonucleotides, where the amount/numberof capture oligonucleotide is typically in excess relative to target).Optionally, the first number of capture oligonucleotide molecules isless than 50%, 25%, 10% or 1% of the number of target nucleic acidmolecules in the sample that are capable of binding to the captureoligonucleotide.

In some embodiments, the capture oligonucleotide binds selectively(e.g., via sequence-specific hybridization) to at least some of thetarget nucleic acid molecules in the sample, creating a population ofbound target nucleic acid molecules. In some embodiments, the methodsfurther include capturing at least some of the bound target nucleic acidmolecules using an agent that selectively binds to the captureoligonucleotide, creating a population of captured target nucleic acidmolecules. In some embodiments, the number of captured target nucleicacid molecules in the population of captured target nucleic acidmolecules is directly proportional to the first number of captureoligonucleotide molecules. This feature can allow the recovery ofsubstantially equal numbers of captured target nucleic acid moleculesfrom multiple samples in parallel, without the need for individuallymeasuring the concentration of such samples.

In some embodiments, the capture oligonucleotide includes a capturemoiety that selectively binds to the binding agent. For example, thecapture moiety can include a first member of a binding pair, and thebinding agent can include a second member of the same binding pair. Insome embodiments, the capture oligonucleotide includes a biotin capturemoiety, and the binding agent is a streptavidin-containing support.

In some embodiments, the disclosure relates generally to methods (andrelated compositions, systems, apparatuses and kits) for isolating aspecific amount of nucleic acid from a sample, comprising: generating apopulation of bound target nucleic acid molecules by contacting a sampleincluding a population of target nucleic acid molecules with a limitingnumber of capture oligonucleotides under conditions where at least someof the capture oligonucleotides hybridize to at least some of the targetnucleic acid molecules. Optionally, the methods further include forminga population of captured target nucleic acid molecules by capturing asubstantial portion of the population of bound target nucleic acidmolecules, wherein the number of captured target nucleic acid moleculesis directly proportional to the limiting number of captureoligonucleotides.

In some embodiments, the disclosure relates generally to a method fornormalizing the concentration of two or more nucleic acid samples,comprising: forming a first population of captured target nucleic acidmolecules from a first sample according to the methods disclosed herein,and forming a second population of captured target nucleic acidmolecules from a second sample according to the same method, wherein thenumber of captured target nucleic acid molecules in the first and secondpopulations of captured target nucleic acid molecules vary no greaterthan 5 fold from each other. In some embodiments, the number of capturedtarget nucleic acid molecules in the first and second populations ofcaptured target nucleic acid molecules varies within a range of about2-fold, 3-fold, or 4-fold.

In some embodiments, the capture oligonucleotide includes a capturesequence that is substantially complementary to a corresponding capturesequence within the target nucleic acid molecule. The Tm of the hybridformed between the capture sequences of the capture oligonucleotide andthe target nucleic acid molecule (referred to herein as “the captureTm”) can be less than about 50° C., 45° C., 40° C., 35° C., 30° C., 25°C., 20° C., or lower.

In some embodiments, the method can further include use of a captureoligonucleotide which can capture or hybridize to the target nucleicacid molecules at permissive conditions, but which does notsignificantly capture or hybridize to the target nucleic acid moleculesat non-permissive conditions. The permissive conditions can include, forexample, lower temperatures (e.g., temperatures significantly below thecapture Tm), high salt (e.g., NaCl concentrations of 0.25M, 0.3M, 0.4M,0.5M, 0.75M, 1M or higher), low or absent levels of denaturingchemicals, etc. The non-permissive conditions can include, for example,higher temperatures (e.g., temperatures significantly above the captureTm), low or absent levels of salt (e.g., NaCl concentrations of 0.2M,0.1M, 0.05M, 0.001M, or low), low or absent levels of denaturingchemicals, etc.

In some embodiments, the disclosed methods can include hybridizing apopulation of target nucleic acid molecules in a sample with a limitingnumber of capture oligonucleotides at permissive conditions to form apopulation of bound target nucleic acid molecules, capturing the boundtarget molecules by capturing the bound target nucleic acid moleculeswith a binding agent to form a population of captured target nucleicacid molecules, and then subjecting the captured nucleic acid moleculesto non-permissive conditions to elute the captured nucleic acidmolecules from the binding agent, thereby forming a population of elutedtarget nucleic acid molecules. The number of eluted target nucleic acidmolecules is directly proportional to the limiting number of captureoligonucleotides used in the hybridizing step. In some embodiments,multiple samples are processed in parallel in this manner using the samenumber of capture oligonucleotides with each sample, and the methodfurther includes recovering substantially equal numbers orconcentrations of eluted target nucleic acid molecules from each sample.

In embodiments involving purification of primer extension products(including primer extension products formed via amplification processessuch as PCR), it can be desirable to avoid unintended capture ofunextended primers by the capture oligonucleotide though use of ahairpin oligonucleotide (also referred to herein as “a hairpin primer”)to drive primer extension. Accordingly, in some embodiments, thedisclosure relates generally to compositions for isolating primerextension products, comprising a hairpin oligonucleotide having a firsthairpin sequence and a second hairpin sequence. In some embodiments, thefirst and/or second hairpin sequences are each independently between 3and 100 nucleotides long, for example between 5 and 20 nucleotides long.In some embodiments, the hairpin oligonucleotide includes a firsthairpin sequence that is substantially or completely complementary to asecond hairpin sequence within the hairpin oligonucleotide. The Tm ofthe hybrid formed between the first and second hairpin sequences(referred to herein as “the hairpin Tm”) can be less than about 50° C.,45° C., 40° C., 35° C., 30° C., 25° C., 20° C., or lower.

In some embodiments, the hairpin primer will exist predominantly inhybridized or hairpin form at permissive conditions, and predominantlyin linear or denatured form at non-permissive conditions. The permissiveconditions can include, for example, lower temperatures (e.g.,temperatures significantly below the hairpin Tm), high salt (e.g., NaClconcentrations of 0.25M, 0.3M, 0.4M, 0.5M, 0.75M, 1M or higher), low orabsent levels of denaturing chemicals, etc. The non-permissiveconditions can include, for example, higher temperatures (e.g.,temperatures significantly above the hairpin Tm), low or absent levelsof salt (e.g., NaCl concentrations of 0.2M, 0.1M, 0.05M, 0.001M, orlow), low or absent levels of denaturing chemicals, etc. In someembodiments, the method can include a permissive condition for hairpinformation and a non-permissive condition for capture targethybridization. In some embodiments, the method can include anon-permissive condition for hairpin formation and a permissivecondition for capture-target hybridization. In some embodiments, themethod can include permissive conditions for both hairpin formation andcapture target hybridization. In some embodiments, the method caninclude non-permissive conditions for both hairpin formation and capturetarget hybridization.

In some embodiments, the disclosed methods can include hybridizing apopulation of target nucleic acid molecules in a sample with the hairpinprimer at nonpermissive conditions to form a population of hairpinprimer-target complexes, and extending the hairpin primer in atemplate-dependent fashion to form a hairpin primer extension product.In some embodiments, the method can further include subjecting theprimer extension mixture to permissive conditions to induce theformation of hairpins by substantially all of the unextended hairpinprimers. This ensures that the unextended hairpin primers do notsignificantly (or substantially) bind to the capture oligonucleotideduring the subsequent capture step.

Optionally, the first and second hairpin sequences of the hairpin primerare separated by a spacer region. The spacer region can optionallyinclude one or more nucleotides or can be comprised entirely ofnon-nucleotidyl moieties. In some embodiments, the spacer regionincludes a non-replicable moiety that cannot be replicated by apolymerase. Such non-replicable moieties can include any moiety thatcannot support template-based nucleotide polymerization by a polymerase.For example, the non-replicable moiety can include a non-nucleotidylmoiety (e.g., PEG or other carbon-based spacer, amino acid, ornucleotide analog that is not recognized by the polymerase used toperform the primer extension, for example uracil in conjunction with aDNA-dependent DNA polymerase, etc). When the hairpin primer is used as atemplate for template-dependent nucleic acid synthesis by a polymerase,the polymerase cannot extend the synthesized nucleic acid strand beyondthe non-replicable moiety. This typically results in the cessation ortermination of nucleic acid synthesis after some portion of the hairpinoligonucleotide has been copied into an opposing strand, leaving theremaining portion of the hairpin oligonucleotide single stranded. Thesynthesized or replicated strand can remain base paired to the hairpinoligonucleotide, forming a hairpin primer extension product that ispartly double stranded and partly single stranded. The single strandedregion optionally includes some portion of the hairpin primer.

In some embodiments, the single stranded region of the hairpin primerextension product includes a capture sequence that can bind to acorresponding capture sequence in the capture oligonucleotide.Optionally, the capture sequence of the hairpin oligonucleotide is atleast 70% complementary to the capture sequence of the captureoligonucleotide. In some embodiments, the capture sequence of thehairpin oligonucleotide and capture oligonucleotides are completelycomplementary to each other.

In some embodiments, the hairpin oligonucleotide is predominantly singlestranded at temperatures significantly above the hairpin meltingtemperature (“hairpin Tm”). At temperatures significantly below thehairpin melting temperature (“hairpin Tm”), the hairpin form of theoligonucleotide predominates.

Optionally, the composition further includes a target nucleic acidmolecule. The target nucleic acid molecule can include a sequence thatis at least partially complementary to a sequence of the hairpin.

Optionally, the composition further includes a capture oligonucleotide.

In some embodiments, the target nucleic acid molecule can include asequence that is at least partially complementary to a sequence of thehairpin.

In some embodiments, the target nucleic acid molecule and the captureoligonucleotide each separately include a sequence that is at leastpartially complementary to the first and/or second hairpin sequences.

In some embodiments, the capture Tm can be selected to allowhybridization of the two capture sequences at permissive conditions,where the two capture sequences are denatured under non-permissiveconditions. The permissive conditions can include any temperaturesignificantly below the capture Tm (e.g., room temperature or below). Insome embodiments, the permissive conditions include high saltconcentrations (e.g., NaCl of 0.1M or higher, typically 0.25M, even moretypically 0.5M or higher). Following capture at permissive conditions,the captured product can optionally be washed to remove non-specificallybound contaminants. The purified primer extension product can then beeluted from the capture oligonucleotide via exposure to non-permissiveconditions (e.g., temperature significantly above capture Tm and/or lowsalt concentrations). Since the amount of capture oligonucleotide usedto perform the capture is limiting, the amount of purified primerextension product will be directly proportional to the number of captureoligonucleotide molecules used to perform the capture. When such captureis performed on multiple samples in parallel using the same number ofcapture oligonucleotides, the number of purified product moleculesobtained from each sample should be directly proportional to the numberof capture oligonucleotide molecules used in the assay. In someembodiments, the number of purified product molecules recovered fromeach sample are substantially equal to each other.

In some embodiments, the disclosure relates generally to kits fornormalization of nucleic acid samples, comprising: a container includinga hairpin oligonucleotide. The hairpin oligonucleotide can include anyhairpin oligonucleotide disclosed herein. In some embodiments, the kitfurther includes the same container (or a different container) includinga capture oligonucleotide. Optionally, the hairpin oligonucleotideincludes a capture sequence that is at least 85% complementary to asequence of the capture oligonucleotide.

In some embodiments, the disclosure relates generally to methods (andrelated compositions, kits, systems and apparatuses) for purifying aprimer extension product from a primer extension reaction mixture,comprising: hybridizing a hairpin primer to a target nucleic acid (or toa population of target nucleic acid molecules) within a sample.Optionally, the hairpin primer includes a first hairpin sequence and asecond hairpin sequence. The first and second hairpin sequences can besubstantially or completely complementary to each other. The Tm of thehybrid formed between the first and second hairpin sequences (“hairpinTm”) can be about 50° C. or lower. The method can optionally includeforming a primer extension reaction mixture including a hairpin primerand one or more target nucleic acid molecules. The method can includehybridizing the hairpin primer to one or more target nucleic acidmolecules of the sample at a nonpermissive temperature at whichsubstantially all (or a significant portion) of the hairpin primers arein linear or extended form. In some embodiments, the method furtherincludes extending the hairpin primer in a target-dependent fashion at apermissive condition to form one or more extended hairpin primerproducts. In some embodiments, the method further includes subjectingthe primer extension reaction mixture including the one or more extendedprimer products to nonpermissive conditions such that substantially all(or a significant portion) of unextended hairpin primers are in thehairpin form. The method can further include contacting the extendedhairpin primers with a capture oligonucleotide under conditionspermitting hybridization of the capture oligonucleotide to the extendedhairpin primers. In some embodiments, the extended hairpin primers arepartly double stranded and partly single stranded, and the captureoligonucleotide hybridizes to a sequence within the single strandedportion of the extended hairpin primers.

In some embodiments, the disclosure relates generally to a method (andrelated compositions and kits) for purifying a primer extension productfrom a primer extension reaction mixture, comprising: hybridizing ahairpin primer to one or more target nucleic acid molecules. Optionally,the hairpin primer includes a first hairpin sequence and a secondhairpin sequence separated by a non-replicable moiety. In someembodiments, the first and second hairpin sequences can be separated bya non-replicable moiety. In some embodiments, the method furtherincludes extending the hairpin primer in a template-dependent fashion toform a hairpin primer extension product having a single stranded regionincluding the first hairpin sequence.

In some embodiments, the method further includes hybridizing the hairpinprimer extension product to a capture oligonucleotide that includes acapture moiety.

In some embodiments, the method further includes selectively capturingthe hairpin primer extension product using a binding agent thatselectively binds to the capture moiety.

In some embodiments, the capture oligonucleotide includes a sequencethat is substantially complementary to at least a portion of the singlestranded region of the hairpin primer extension product.

In some embodiments, the capture oligonucleotide includes a sequencethat is substantially complementary to at least a portion of the firsthairpin sequence.

In some embodiments, the extending is performed at a temperature(“extension temperature”) that is significantly above the hairpin Tm,such that the first and second hairpin sequences of the hairpin primerdo not substantially hybridize to each other at the extensiontemperature.

In some embodiments, the primer extension product is hybridized to thecapture oligonucleotide at a temperature (“capture temperature”)significantly below the hairpin Tm, such that substantially allunextended hairpin oligonucleotide is in the hairpin form at the capturetemperature.

In some embodiments, the disclosure relates generally to kits forisolating target nucleic acid molecules using a capture oligonucleotide,as well as related compositions, methods, systems and apparatuses. Insome embodiments, the kits (and relating compositions, systems,apparatuses and methods) include a capture oligonucleotide capable ofselectively binding to one or some portion of members of a population oftarget nucleic acid molecules. In some embodiments, the kit canoptionally include the capture oligonucleotide in a limited amount oramount that can be diluted so as to be present in a limiting number,compared to the population of target nucleic acid molecules that canselectively bind to the capture oligonucleotide. In some embodiments,the capture oligonucleotide can be present in an amount less than 50%,25%, 20%, 10%, 5% or 1% of the number of target nucleic acid moleculesin the sample capable of binding to the capture oligonucleotide. In someembodiments, the capture oligonucleotide includes a capture moiety thatselectively binds to a binding agent. For example, the capture moietycan include a first member of a binding pair, and the binding agent caninclude a second member if the same binding pair. In some embodiments,the kit can further include one or more hairpin primers. The hairpinprimers can include any hairpin primer disclosed herein. In someembodiments, the hairpin primer includes a first hairpin sequence and asecond hairpin sequence that are complementary to each other and willhybridize to each other (forming a hairpin structure) at temperaturessignificantly below the hairpin Tm. Optionally, the kit can furtherinclude a binding agent that selectively binds to the captureoligonucleotide.

EXAMPLES

FIG. 1 depicts an exemplary embodiment of a normalization method asdescribed in Example 1, wherein a target nucleic acid molecule isamplified using a hairpin primer (for forward strand synthesis) and areverse primer (for reverse strand synthesis) using PCR. The hairpinprimer includes a first hairpin sequence and a second hairpin sequencethat are complementary to each other and will hybridize to each other atpermissive temperatures (e.g., temperatures significantly below thehairpin Tm) and/or at permissive salt concentrations. The hairpin primeralso includes a non-replicable spacer comprising a carbon-18 (C18)spacer. The amplification product includes the hairpin oligonucleotide,part of which remains single stranded. The single stranded region of theamplification product includes a capture sequence, which iscomplementary to a corresponding capture sequence on a biotinylatedcapture oligonucleotide. The capture sequence has an estimated Tm ofabout 36° C. in 50 mM NaCl. The amplified product is captured byhybridization to a limiting amount of capture oligonucleotide (133 pM in75 ul) at permissive temperatures (e.g., room temperature or lower)and/or permissive salt concentrations (e.g., 0.5M NaCl or greater). Thecapture oligonucleotide-product complex can then be selectively capturedusing the binding agent streptavidin, e.g., via mixture withstreptavidin-coated beads. The beads containing the captured product canbe washed to remove non-specifically bound material. The capturedamplified product can be eluted from the beads by using an elutionsolution including non-permissive (low) salt concentrations and/orexposure to non-permissive (elevated) temperatures to denature thehybridization product between the capture sequence of the captureoligonucleotide and the capture sequence of the single stranded portionof the amplified product.

The target enrichment methods disclosed herein can be advantageouslyused to simplify the workflow and reduce the cost and effort ofpreparing large numbers of samples for analysis by allowing thepreparation of large numbers of nucleic acid samples containingsubstantially equal numbers (or concentrations) of nucleic acidmolecules.

Example 1: Library Normalization by Limiting Capture

FIGS. 2 and 3 are representative data obtained from 10 DNA samples (2matched lung normal/tumor pairs, 3 formalin-fixed paraffin embedded DNAsamples (FFPE), and 3 high molecular weight DNAs) that were carriedthrough a 207-plex amplicon library preparation workflow using 5 ng, 10ng, and 20 ng of DNA input. Libraries were prepared in triplicate foreach sample at each input level.

Sequencing of these nucleic acid libraries showed no adverse effects onaccuracy (>99.5%), uniformity of amplicon representation (>99%), orbases with no strand bias (>99%).

Methods and Materials

Ten human DNA samples (2 matched lung normal/tumor pairs, 3 FFPEsamples, and 3 high molecular weight reference DNAs) were carriedthrough the Ion AmpliSeq 2.0 Library Preparation workflow (Ion Ampliseq™Library Preparation, Publication Part Number MAN0006735, LifeTechnologies, CA) using the Ion Ampliseq™ Cancer Hotspot Panel v2 (LifeTechnologies, CA, Cat. No. 4475346) starting with 5, 10, and 20 ng foreach DNA sample. The Ion Ampliseq™ Cancer Hotspot Panel is a 207-plexcancer panel primer pool used to perform multiplex PCR for preparationof amplicon libraries from genomic “hotspot” regions that are frequentlymutated in human cancer genes.

Briefly, the workflow was as follows. All temperature incubations werein a PCR thermocycler (except for room temperature). Reagents were fromthe Ion AmpliSeq™ Library Kit 2.0 (Life Technologies, CA, Cat. No.4480441), unless otherwise noted.

Amplicons were produced by “pre-amplification” in 20 μL reactionsconsisting of 1× Ion AmpliSeq™ HiFi Master Mix (sold as a component ofthe Ion Ampliseq™ Library Kit 2.0, Cat. No. 4480441), 1× Ion Ampliseq™Cancer Hotspot Panel v2 primers (Life Technologies, CA, Cat. No.4475346), and either 5 ng, 10 ng, or 20 ng human genomic DNA. Each DNAsample was pre-amplified in triplicate at each input level. Reactionswere distributed to individual wells of a 96 well PCR plate (MicroAmp®Optical 96-Well Reaction Plate, Life Technologies, CA, Cat. No.N8010560), the plate sealed and incubated as follows: 2 minutes at 99°C., then 20 cycles (for FFPE samples) or 17 cycles of 15 seconds at 99°C. and 4 minutes at 60° C., after which the thermocycler was held at 10°C.

Amplicons from pre-amplification were prepared for ligation by treatmentwith Ion FuPa Reagent (2 μL blend/well)(sold as a component of the IonAmpliseq™ Library Kit 2.0, Cat. No. 4480441) for 10 minutes at 50° C.,10 minutes at 55° C., 10 minutes at 60° C., then held at 10° C.

Ligation of sequencing adapters was achieved by addition of 4 μL SwitchSolution (sold as a component of the Ion Ampliseq™ Library Kit 2.0, Cat.No. 4480441), 2 μL Ion Ampliseq™ Adapters (sold as a component of theIon Ampliseq™ Library Kit 2.0, Cat. No. 4480441), and 2 μL DNA ligase(sold as a component of the Ion Ampliseq™ Library Kit 2.0, Cat. No.4480441) to each well, followed by incubation for 30 minutes at 22° C.,10 minutes at 72°, then held at 10° C.

Ligated amplicons were purified by addition of 45 μL (1.5× volume)AMPure XP Kit (Beckman Coulter, Cat. No. A63880) to each well,incubation for 5 min at room temperature, then drawing the AMPure beadsinto pellets by placing the PCR plate on a plate magnet (DynaMag™-96Side, Life Technologies, Cat. No. 12331D) for 2 minutes.

Supernatants were removed by pipet and 150 μL of 70% ethanol (v/v inwater) was added to each well. Pellets were washed by toggling the plateposition two times in the magnet to move pellets from side to side inthe wells. Wash was removed by pipet and the washing was repeated asecond time for each well. Following removal of the second wash, thepellets were allowed to dry for 5 minutes at room temperature, thenre-suspended in 50 μL Library Amplification Primer Mix (a mixture ofPlatinum PCR SuperMix High Fidelity (sold as a component of the IonAmpliseq™ Library Kit 2.0, Cat. No. 4480441) plus Equalizer primers(provided at a concentration of 400 pM).

The plate was returned to the magnet for 2 minutes, the supernatantswere removed to clean the wells, the plate was sealed and incubated for2 minutes at 98° C., followed by eight cycles of 15 seconds at 98° C.and 1 minute at 60° C., and then held at 10° C. An aliquot (2 μL) ofeach amplified nucleic acid library was diluted for in-processquantitation by qPCR using the Ion Library Quantitation Kit (LifeTechnologies, CA, Cat. No. 4468802) according to the instructions.

Amplified libraries were normalized directly out of the libraryamplification process described above, without purification, by additionof 25 μL Equalizer Capture oligonucleotide (final concentration 133 pM),incubation for 5 minutes at room temperature, addition of 5 μL DynaBeadsMyOne streptavidin Cl beads (sold as a component of the Ion Ampliseq™Library Kit 2.0, Cat. No. 4480441)(washed and re-suspended in twovolumes Equalizer Wash Buffer (10 mM Tris pH 8, 500 mM NaCl, 0.1M EDTA,0.05% Tween-20), incubation for 5 minutes at room temperature, followedby pelleting the DynaBeads on a plate magnet as described above.Supernatants were removed by pipet and 150 μL/well Equalizer Wash Bufferwas added to each well. Pellets were washed by toggling the plateposition three times in the magnet to move pellets from side to side inthe wells. Wash was removed by pipet and the washing was repeated asecond time for each well. Following removal of the second wash, thepellets were re-suspended in 50 μL Equalizer Elution Buffer (10 mM TrispH 8, 0.1 mM EDTA and 0.1 μg/μL glycogen) and the plate was returned tothe magnet for 2 minutes. The supernatants containing normalizedlibraries were removed to clean wells. Aliquots of each library werediluted for quantitation by qPCR using the Ion Library Quantitation Kit(Life Technologies, CA, Cat. No. 4468802) according to themanufacturer's instructions.

Results:

Quantitation of Libraries by qPCR Prior to Normalization.

Aliquots of all libraries were diluted 5,000-fold and assayed intriplicate by qPCR with the Ion Library Quantitation Kit (LifeTechnologies, CA, Cat. No. 4468802) according to the manufacturer'sinstructions. Total library yields ranged from 4-94 nM after libraryamplification and prior to normalization (FIG. 2).

Quantitation of Libraries by qPCR After Normalization.

Aliquots of all libraries were diluted 100-fold and assayed intriplicate by qPCR with the Ion Library Quantitation Kit (LifeTechnologies, CA, Cat. No. 4468802) according to the manufacturer'sinstructions. Final library yields after normalization ranged from33-204 pM, with 84/90 (93%) libraries within 701-200 pM (FIG. 3).

What is claimed:
 1. A method for normalizing a desired amount of nucleicacid from a sample, comprising: a) generating a population of boundtarget nucleic acid molecules by contacting a sample including apopulation of target nucleic acid molecules with a hairpin primer whichinclude a first primer sequence and a second primer sequence separatedby a non-replicable moiety, wherein the second primer sequence binds aportion of the target nucleic acid molecules, and wherein the firstprimer sequence binds a capture oligonucleotide, under conditions whereat least some of the hairpin primers hybridize to at least some of thetarget nucleic acid molecules; and b) forming a population of capturedtarget nucleic acid molecules by contacting the bound target nucleicacid molecules with a limiting, desired amount of the captureoligonucleotides, wherein the capture oligonucleotide includes a capturemoiety, thereby capturing a desired portion of the population of boundtarget nucleic acid molecules, wherein the number of captured targetnucleic acid molecules is directly proportional to the limiting numberof capture oligonucleotides.
 2. A method for normalizing theconcentration of two or more nucleic acid samples, comprising: forming afirst population of captured target nucleic acid molecules from a firstsample according to the method of claim 1, and forming a secondpopulation of captured target nucleic acid molecules from a secondsample according to the method of claim 1, wherein the number ofcaptured target nucleic acid molecules in the first and secondpopulations of captured target nucleic acid molecules vary by no greaterthan 5-fold from each other.
 3. The method of claim 1, wherein thecapture moiety comprises a biotin moiety.
 4. The method of claim 1,wherein the population of bound target nucleic acid molecules iscaptured by contacting the bound target nucleic acid molecules with abinding agent.
 5. The method of claim 4, wherein the binding agentcomprises a streptavidin.
 6. The method of claim 4, wherein the bindingagent includes a support.
 7. The method of claim 6, wherein the supportcomprises a bead.
 8. The method of claim 1, further comprisingsubjecting the captured nucleic acid molecules to non-permissiveconditions to elute the captured nucleic acid molecules from the bindingagent, thereby forming a population of eluted target nucleic acidmolecules.
 9. The method of claim 1 wherein the first and second primersequences of the hairpin primer are substantially complementary to eachother and have a first Tm.
 10. The method of claim 9 further comprisingextending the hairpin primer in a template-dependent fashion to form aprimer extension product having a single stranded region including thefirst primer sequence before forming the population of captured targetnucleic acid molecules.
 11. The method of claim 10 wherein the captureoligonucleotide includes a sequence that is substantially complementaryto at least a portion of the single stranded region of the primerextension product.
 12. The method of claim 10 wherein the extending isperformed at a temperature that is significantly above the first Tm,such that the first and second primer sequences of the hairpin primer donot substantially hybridize to each other.
 13. The method of claim 10wherein the primer extension product is hybridized to the captureoligonucleotide at a temperature significantly below the first Tm, suchthat substantially all unextended hairpin oligonucleotide is in thehairpin form.
 14. The method of claim 10 wherein hybridizing the primerextension product to a capture oligonucleotide that includes a capturemoiety and selectively capturing the primer extension product uses abinding agent that selectively binds to the capture moiety.
 15. A methodfor normalizing a desired amount of nucleic acid from a sample,comprising: a) generating a population of bound target nucleic acidmolecules by contacting a sample including a population of targetnucleic acid molecules with a hairpin primer which include a firstprimer sequence and a second primer sequence separated by anon-replicable moiety, wherein the second primer sequence binds aportion of the target nucleic acid molecules, and wherein the firstprimer sequence binds a capture oligonucleotide, under conditions whereat least some of the hairpin primers hybridize to at least some of thetarget nucleic acid molecules; b) forming a population of capturedtarget nucleic acid molecules by contacting the bound target nucleicacid molecules with a limiting, desired amount of the captureoligonucleotides and a binding agent, wherein the captureoligonucleotide includes a capture moiety and the capture moiety bindsthe binding agent, thereby capturing a desired portion of the populationof bound target nucleic acid molecules, wherein the number of capturedtarget nucleic acid molecules is directly proportional to the limitingnumber of capture oligonucleotides; and c) subjecting the capturednucleic acid molecules to non-permissive conditions to elute thecaptured nucleic acid molecules from the binding agent, thereby forminga normalized population of target nucleic acid molecules.
 16. The methodof claim 15 further comprising extending the hairpin primer in atemplate-dependent fashion to form a primer extension product having asingle stranded region including the first primer sequence beforeforming the population of captured target nucleic acid molecules. 17.The method of claim 16 wherein the extending is performed at atemperature that is significantly above the Tm of the hairpin primer,such that the first and second primer sequences of the hairpin primer donot substantially hybridize to each other.
 18. The method of claim 16wherein the primer extension product is hybridized to the captureoligonucleotide at a temperature significantly below the Tm of thehairpin primer, such that substantially all unextended hairpin primer isin the hairpin form.
 19. The method of claim 16 wherein hybridizing theprimer extension product to the capture oligonucleotide that includesthe capture moiety and selectively capturing the primer extensionproduct uses the binding agent that selectively binds to the capturemoiety.
 20. A method for normalizing the concentration of two or morenucleic acid samples, comprising: forming a first population of capturedtarget nucleic acid molecules from a first sample according to themethod of claim 16; and forming a second population of captured targetnucleic acid molecules from a second sample according to the method ofclaim 16, wherein the number of captured target nucleic acid moleculesin the first and second populations of captured target nucleic acidmolecules vary by no greater than 5-fold from each other; and optionallycombining the populations of two or more captured target nucleic acidmolecules.